Mitochondrial content and overall electron transport chain activity are severely depressed (50-70%) in skeletal muscle of obese arid obese/diabetic patients. These data imply that obesity, or more likely the over nutrition that causes obesity, leads to a progressive decline in mitochondrial function, eventually culminating in the dissolution and loss of mitochondria. Mitochondrial dysfunction has also been implicated in the etiology of insulin resistance;however, the underlying mechanisms leading to mitochondrial dysfunction and their potential link to the development of insulin resistance, particularly in the context of obesity, are unknown. The long term objective of this research is to determine if the loss of mitochondrial integrity and insulin sensitivity stem from a common metabolic disturbance, i.e., oxidative stress. Our hypothesis is that over nutrition, particularly from high fat diets, dramatically increases the propensity for mitochondrial reactive oxygen species (ROS) emission in skeletal muscle, leading to both mitochondrial dysfunction and the development of insulin resistance. The specific aims of this project are designed to 1) determine the mechanisms responsible for the loss of mitochondrial function induced by a high fat diet, and 2) determine if mitochondrial-derived oxidative stress is a primary factor linking over nutrition to the loss of mitochondrial function and development of insulin resistance in skeletal muscle. The project will utilize a newly developed permeabilized fiber approach that preserves the natural reticular structure of mitochondria in skeletal myofibers, and state of the art pharmacological agents and transgenic models to manipulate mitochondrial ROS production and scavenging. Relevance to Public Health. Achieving the aims of this application will establish mitochondrial derived oxidative stress as a primary cause of diet-induced mitochondrial dysfunction and insulin resistance in skeletal muscle. This will fundamentally alter the context in which metabolic imbalance is viewed, i.e., how cells regulate and govern energy balance in real-time, and will therefore provide a mechanistic link between mitochondrial bioenergetics and the factors known to cause (over nutrition, sedentary lifestyle), prevent (metabolic balance, physical activity), and treat (caloric restriction, increased physical activity) insulin resistance and type II diabetes.